p53 tumor suppressor is a transcription factor and can be activated in response to various stress signals, including DNA damage, oncogene activation, and hypoxia. Upon activation, p53 exerts its tumor suppression by regulating a plethora of genes involved in the control of the cell cycle, apoptosis, DNA repair and other cellular processes. p21, a cyclin-dependent kinase inhibitor, is transcriptionally regulated by the p53 family to induce cell cycle arrest. p21 is also regulated post-transcriptionally upon DNA damage in a p53-dependent manner, but the mechanism is uncertain. During the last funding period, we identified several novel genes, which can be induced by p53 and DNA damage in a p53-dependent manner. Among these is the RNPC1 gene, which encodes at least two alternative spliced isoforms, RNPC1a and RNPC1b, both of which contain an intact RNA recognition motif. We found that over-expression of RNPC1a, but not RNPC1b, is capable of inducing cell cycle arrest in G1. We also found that while both isoforms directly bind to the 3'-untranslated region in p21 transcript, only RNPC1a is able to stabilize both the basal and stress-induced p21 transcripts. Conversely, RNPC1a knockdown decreases the basal level of p21 transcript. Thus, Specific Aim 1 is proposed to test the hypothesis that an mRNA decay pathway is regulated by RNPC1 to modulate p21 mRNA stability. The activity of p53 is primarily controlled by post-transcriptional mechanisms. While it is well known that modifications of p53, such as phosphorylation and acetylation, stabilize and activate p53, increasing evidence indicate that other post-transcriptional mechanisms play a major role in modulating p53 activity. For example, ribosomal protein L26 and nucleolin are found to bind to p53 transcript and positively and negatively regulate p53 translation, respectively. In addition, HuR is found to enhance p53 translation as well as p53 mRNA stability. Interestingly, HuR is also found to bind to p21 transcript and regulate p21 mRNA stability. As a RNA binding protein and a target of p53, we found that RNPC1 binds to p53 transcript and knockdown of total RNPC1 or RNPC1a increases the basal levels of p53 and further enhances p53 accumulation induced by DNA damage. Conversely, we found that over-expression of RNPC1a, but not RNPC1b, inhibits the basal levels of p53 and DNA damage-induced p53 accumulation. These data suggest that p53 is likely to be regulated by RNPC1. Furthermore, recent studies demonstrated that p53 translation is increased under stress conditions and our preliminary studies indicate that the efficacy of p53 translation is modulated by RNPC1 under both normal and stress conditions. Thus, Specific Aim 2 is proposed to test the hypothesis that RNPC1 regulates the cap-dependent translation machinery to modulate p53 translation under normal conditions and the cap-independent translation machinery to modulate p53 translation under stress conditions. PUBLIC HEALTH RELEVANCE: Regulation of p53 activity has always been at the center of p53 research as the majority of human cancers have a defective p53 or a faulty p53 pathway. The proposed study to analyze how RNPC1, as a p53 target gene, regulates p53 is highly significant in several areas. First, it will further our understanding of the p53 biology, which is needed in order to explore the p53 pathway for cancer diagnosis and treatment. Second, RNPC1 may be explored as a molecule target to activate p53 to sensitize tumor cells which contain wild-type p53, or to repress wild-type p53 to de-sensitize normal cells, to radiation- and chemo-therapies. Third, as a regulator of p53 translation, RNPC1 would regulate expression of mutant p53 in tumor cells. Since mutant p53 is an oncogene, RNPC1 may be explored to repress mutant p53 and thus, inhibit tumor growth and/or sensitize tumor cells to radiation- and chemo-therapies. Due to loss of p53 and other factors that are required for p21 expression, tumor cells are often dysfunctional in the cell cycle control, in which p21 is a key player. Restoration of proper cell cycle control in tumor cells is one of the many approaches that have been explored to suppress cell transformation and tumor progression and metastasis. We showed that p21 mRNA stability is regulated by RNPC1 under both normal and stress conditions. Thus, the proposed study to analyze how p21 expression is regulated by RNPC1 will further our understanding whether a cell cycle control mechanism can be restored in tumor cells.